PROJECT SUMMARY/ABSTRACT Nonalcoholic fatty liver disease (NAFLD) results from excessive accumulation of fat in the liver (steatosis). A subset of NAFLD patients will also develop steatohepatitis, fibrosis, and cirrhosis, which is associated with a heightened risk for liver cirrhosis and increased liver-related morbidity and mortality. Clinical outcomes for NAFLD patients with coincident hepatocyte injury and liver inflammation are substantially worse compared to individuals with simple steatosis, yet to date, clinical characteristics and laboratory values have not been useful for predicting disease severity in NAFLD. Further, the molecular mechanisms underlying the heterogeneous outcomes of NAFLD remain poorly understood, which limits accurate diagnosis and treatment of the disease. An important clinical challenge, therefore, is to distinguish those patients with NAFLD who are more likely to develop clinically severe forms of fatty liver disease from those who will not. Emerging evidence supports a role for epigenetic factors, particularly DNA methylation, in the development of NAFLD, which may link environmental exposures with pathophysiological mechanisms. The overall goal for this project is to characterize the role of DNA methylation in the development of NAFLD fibrosis. The specific aims of this study are to first perform DNA methylation profiling to identify CpG sites that are differentially methylated between NAFLD without fibrosis and NAFLD with fibrosis using linear mixed effects regression. We will follow up on the most differentially methylated CpG loci in an independent sample of individuals from the same cohort as well as participants from the NASH Clinical Research Network (CRN) using the same design as the discovery cohort matched for age, sex, and ethnicity. We will then integrate data from high throughput RNA sequencing of liver biopsies from the discovery cohort with DNA methylation results to identify genes that are coordinately affected by methylation status and associated with the presence of fibrosis. Finally, we will combine already acquired data from genome-wide genotyping and results from the DNA methylation analyses to identify cis methylation quantitative trait loci (meQTLs). Characterization of the key sites regulated by DNA methylation and the associated effects on gene expression will improve our understanding of the biology underlying NAFLD-related fibrosis. Such information may lead to the identification of novel targets for therapeutic intervention and/or improved methods for identifying NAFLD patients who will likely develop coincident fibrosis and/or cirrhosis. Given the substantial public health burden of NAFLD, which is increasing at alarming rates due to the rising prevalence of obesity, novel therapeutic targets are urgently needed to facilitate the development of improved pharmacological therapies for the treatment and prevention of the disease.